DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of the Application
Claim(s) 1-4, 6-7, 12-13, 17, 21, 23 are pending and under examination
Claim(s) 25-26, 31-33, 38, 43, 47, 49, and 51 are withdrawn from consideration.
Applicant’s election without traverse in the reply filed on 03/26/2026 of (A) the method exemplified in claim 1; (B), the calculation exemplified in claim 12; (C) cell-free DNA (cfDNA); (D), Enriching for fragments of a predetermined length; and (E) nucleic acid units expressed in microliters, nanograms, and moles is acknowledged.
Claim(s) 25, 26, 31-33, 38, 43, 47, 49, and 51 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Claims 25, 26, 31-33, 38, 43, 47, and 49 are directed to nonelected method exemplified by claim 25, and claim 51 is directed to the nonelected method exemplified by claim 51. Although these claims may read on the elected features under categories B-E, they do not read on the elected method under category A and hence do not read on the complete elected species.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1-4, 6-7, 12-13, 17, 21, 23 is/are rejected under 35 U.S.C. 103 as being unpatentable over Filippova et al. (US20190287649A1, EFD: March 13th 2018, disclosed in IDS) in view of Mayday et al. (PloS one 14.1 (2019)).
Regarding claim 1, Filippova discloses method comprising the steps of: a. isolating and purifying nucleic acid from a plurality of test subjects to generate corresponding samples of origin to generate at least one sample of origin (e.g. biological sample are obtained from a subject’s blood or plasma containing cfDNA. The cfDNA can be isolated using commercial cell-free DNA isolation kits [¶0287-0290]).
b. preparing a library for each test subject wherein the nucleic acid fragments are barcoded and wherein each library corresponds to a specific sample of origin (e.g. During library preparation, unique molecular identifiers (UMIs) are added to the nucleic acid molecules (e.g., DNA molecules). In some embodiments, e.g., when multiplex sequencing will be used to sequence cfDNA from a plurality of subjects in a single sequencing reaction, a patient-specific index is also added to the nucleic acid molecules [¶0291].)
c. adding a first number of nucleic acid units from each sample of origin to form a first pooled test sample (e.g. cfDNA sequencing libraries are pooled together [¶0293])
d. determining the fragment size distribution within each sample of origin (e.g. determining the distribution of cell-free DNA fragment lengths, as determined by whole genome sequencing (WGS) [¶0305-0306, 0335].)
e. determining the abundance of a target nucleic acid population in each sample of origin (e.g. For each patient, estimate the proportion of sequence reads originate from tumor-derived cfDNA [¶0335 and 0347])
h. performing fragment size selection on the second pooled test sample and isolating the target nucleic acid population in suspension to form a third pooled test sample enriched for said target nucleic acid population, wherein said third pooled test sample is ready for diagnostic assay. (e.g. Filippova teaches pooling cfDNA libraries, then size selecting a pool of cfDNA libraries in a single reaction (e.g., a single well of an agarose-based electrophoretic technique) [¶0293-0294]. This isolates shorter fragments and enrich the cancer derived cfDNA [¶0295, 0344, 0347]. Since the enriched fragments are recovered for sequencing and cancer classification, the resulting sample corresponds to the claimed third pooled test sample containing the target population in suspension and ready for diagnostic assay [¶0299-0302]
Filippova does not disclose (f) calculating a unique numerical offset value for each sample of origin and (g) adding a second number of nucleic acid units from each sample of origin based on the unique numerical offset value to form a second pooled test sample.
Mayday discloses a two-step process to optimize the pooling of hundreds of samples. First, dispense equal volumes (500nL) from each sample of a set of 265 libraries to an initial pool. This pool then sequenced, assigned the resulting reads to each barcoded library, calculated the percentage of total reads attributable to each sample, and estimated each sample’s partial concentration. Mayday uses those sample specific ratios and concentrations to calculate an equimolar dispensing volume for each library and dispense calculated volumes ranging from 160nL to 3800nL to form a final balanced pool [“Improving high-throughput library pooling” section]. In another word, Mayday’s equal 500nL input from each library corresponds to the claimed first number of nucleic acid and first pooled test. Mayday’s sample specific read ratio or and/or estimated partial concentration corresponds to the claimed unique numerical offset value. Mayday’s calculated 160-3,800nl dispensing volume for each library corresponds to the claimed second number of nucleic acids to form second pooled sample test.
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to modify Filippova’s patient indexed cfDNA pooling and size selection method by adding Mayday’s preliminary sequencing and calculated re-pooling procedure before Filippova’s pooled size selection step because Mayday explicitly teaches the optimization method helps overcome traditional pooling methods’ drawbacks, which are costly, tedious, and error-prone due to imprecise estimations and inaccurate pipetting, especially when pooling large numbers of libraries together [“Improving high-throughput library pooling” section]. Although Mayday uses RNA libraries as an example, its rebalancing method works on barcoded libraries regardless of whether they originated from RNA or cfDNA since the composition remains nucleic acids. Since both references uses barcoded pooled libraries and pooled sequencing, a skilled artisan would have expected Mayday’s method to work with Filippova‘s cfDNA libraries. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Regarding claim 2, Filippova further discloses the step of sequencing said third pooled test sample and screening the target nucleic acid population for genetic anomalies. (e.g. sequencing the size selected cfDNA pool and analyzing results to detect cancer related genetic abnormalities, including somatic variants and copy number aberrations [¶0244-0248, 0276-0279, 0299, and 0333])
Regarding claims 3 and 4, Filippova further discloses fragment size distribution is determined by paired-end sequencing (e.g. cfDNA fragment size distribution using whole genome sequencing [¶0306 and 0335]. “sequence reads” produced by paired-end sequencing for cfDNA libraries [¶0104 and 0299]).
Regarding claim 6, Filippova further discloses pairing the nucleic acid fragments in the third pooled test sample with the respective sample of origin. (e.g. In some embodiments, the patient specific index are added to ends of DNA fragments during library construction, that serve as a unique tag that can be used to identify sequence reads originating from a specific patient sample. [¶0291 and 0299]).
Regarding claim 7, Filippova further discloses nucleic acid is selected from genomic DNA, FFPE DNA, RNA or cell-free DNA, or wherein said nucleic acid is isolated from whole blood. (e.g. biological sample are obtained from a subject’s blood or plasma containing cfDNA. The cfDNA can be isolated using commercial cell-free DNA isolation kits [¶0287-0290]).
Regarding claim 12, Mayday discloses dispense equal volumes (500nL) from each sample, determine each library abundance from its barcode assigned reads, and estimate sample partial concentration to calculate second pooling volume [“Improving high-throughput library pooling” section]. Since concentration is the amount of material per unit volume, it would have been prima facie obvious to a person of ordinary skill in the art to calculate each sample numerical offset by dividing its target population abundance by the known 500nL input volume. This calculation provides the relative target concentration needed to determine amount of each library added to the second pool.
Regarding claim 13, Filippova further discloses target nucleic acid population is a tumor fraction of a cell free DNA (e.g. the source of copy number events derived from cfDNA can be any one of a germline source, somatic non-tumor source, or somatic tumor source [¶0245, 0335, 0344-0347]).
Regarding claim 17, Filippova further discloses target nucleic acid population is enriched for fragments within a predetermined length range (e.g. cfDNA molecules are size selected within threshold length ranges, including 30-140 and 30-150 nucleotides, to enrich for cancer derived cfDNA [¶0294-0298 and 0344-0347])
Regarding claim 21, Mayday further discloses wherein said first and second number of nucleic acid units is selected from the group consisting of microliters, nanograms, and moles, (e.g. dispense equal volumes (0.5 m µL) from each sample to an initial pool. This pool then sequenced and estimated each sample’s partial concentration. Mayday uses those sample specific ratios and concentrations to calculate an equimolar dispensing volume for each library and dispense calculated volumes ranging from 0.160 µL to 3.800 µL to form a final balanced pool [“Improving high-throughput library pooling” section].)
Regarding claim 23, Filippova further discloses the step of performing whole genome sequencing. [¶0228, 0231, 0235, 0245, 0335-0336, 0347]
Conclusion
No claims are allowed
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Khai Quynh Tien Pham whose telephone number is (571)272-6998. The examiner can normally be reached M-T, 9-4 ET.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached at (571) 272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/KHAI QUYNH TIEN PHAM/ Examiner, Art Unit 1684
/JEREMY C FLINDERS/ Primary Examiner, Art Unit 1684
Prosecution Insights